MRGPRX2 and Immediate Drug Hypersensitivity: Insights From Cultured Human Mast Cells.

BACKGROUND AND OBJECTIVES: Mast cell (MC) degranulation via activation of the Mas-related G protein-coupled receptor X2 (MRGPRX2) plays a key role in immediate drug hypersensitivity (IDH). However, data in humans are limited to observations in specific cell lines. Objective: To study the usefulness of silencing MRGPRX2 in human MCs with the aim of further unveiling the MRGPRX2 pathway in IDH. METHODS: MCs were cultured from CD34+ progenitor cells obtained from peripheral blood (PBCMCs) and incubated with substance P (as a positive control), rocuronium, moxifloxacin, morphine, or amoxicillin. Immunophenotyping of the cells included flow cytometry and microscopy analyses of the expression of CD117, CD203c, and MRGPRX2. Intracellular calcium was measured using Fluo-4. Degranulation was analyzed by quantifying CD63 expression. For MRGPRX2 silencing, MCs were electroporated with Dicer small interference RNAs. RESULTS: Incubation of MCs with substance P, morphine, and moxifloxacin increased intracellular calcium levels and triggered MC degranulation, which, for the drugs, is almost completely abolished by selective MRGPRX2 silencing. Despite an increase in intracellular calcium in MRGPRX2+ cells, incubation with nontoxic concentrations of rocuronium does not result in degranulation of PBCMCs. Amoxicillin has no effect on PBCMCs. CONCLUSION: The use of MRGPRX2 silencing in human MCs can provide important insights into the role of MRGPRX2 in the pathogenesis of IDH. As induction of calcium signals does not necessarily translate into a secretory response, measurement of the degranulation reaction seems more meaningful in the context of drug testing.

MRGPRX2 and Immediate Drug Hypersensitivity: Insights From Cultured Human Mast Cells

Background: Mast cell (MC) degranulation via activation of the Mas-related G protein–coupled receptor X2 (MRGPRX2) plays a key role in immediate drug hypersensitivity (IDH). However, data in humans are limited to observations in specific cell lines. Objective: To study the usefulness of silencing MRGPRX2 in human MCs with the aim of further unveiling the MRGPRX2 pathway in IDH. Methods: MCs were cultured from CD34+ progenitor cells obtained from peripheral blood (PBCMCs) and incubated with substance P (as a positive control), rocuronium, moxifloxacin, morphine, or amoxicillin. Immunophenotyping of the cells included flow cytometry and microscopy analyses of the expression of CD117, CD203c, and MRGPRX2. Intracellular calcium was measured using Fluo-4. Degranulation was analyzed by quantifying CD63 expression. For MRGPRX2 silencing, MCs were electroporated with Dicer small interference RNAs. Results: Incubation of MCs with substance P, morphine, and moxifloxacin increased intracellular calcium levels and triggered MC degranulation, which, for the drugs, is almost completely abolished by selective MRGPRX2 silencing. Despite an increase in intracellular calcium in MRGPRX2+ cells, incubation with nontoxic concentrations of rocuronium does not result in degranulation of PBCMCs. Amoxicillin has no effect on PBCMCs. Conclusion: The use of MRGPRX2 silencing in human MCs can provide important insights into the role of MRGPRX2 in the pathogenesis of IDH. As induction of calcium signals does not necessarily translate into a secretory response, measurement of the degranulation reaction seems more meaningful in the context of drug testing (AU)

Antecedentes: La desgranulación de los mastocitos (MC) a través de la activación del receptor X2 acoplado a proteína G relacionada con Mas (MRGPRX2) se considera clave para la hipersensibilidad inmediata a fármacos. Sin embargo, los datos en humanos se limitan a observaciones en líneas celulares específicas. Objetivo: Estudiar la utilidad del silenciamiento de MRGPRX2 en MC humanos para conocer mejor la vía MRGPRX2 en la hipersensibilidad inmediata a fármacos. Métodos Los MC se cultivaron a partir de células progenitoras CD34+ obtenidas de sangre periférica (PBCMC) y se incubaron con la sustancia P como control positivo, rocuronio, moxifloxacina, morfina o amoxicilina. El inmunofenotipaje de las células incluyó análisis por citometría de flujo y microscopia de la expresión de CD117, CD203c y MRGPRX2. El calcio intracelular se midió usando Fluo-4. La desgranulación se analizó por cuantificación de la expresión de CD63. Para el silenciamiento de MRGPRX2, los MC se electroporaron con ARN silente del sustrato Dicer. Resultados: La incubación de MC con sustancia P, morfina y moxifloxacina provocó el aumento de los niveles de calcio intracelular y desencadenó la desgranulación de MC. En el caso de la desgranulación provocada por los fármacos, ésta se eliminó casi por completo mediante el silenciamiento selectivo de MRGPRX2. A pesar del aumento del calcio intracelular en las células MRGPRX2+, la incubación con concentraciones no tóxicas de rocuronio no produce la desgranulación de los PBCMC, mientras que la amoxicilina no tiene efecto sobre los PBCMC. Conclusión: El uso del silenciamiento de MRGPRX2 en MC humanos puede proporcionar información importante sobre el papel de MRGPRX2 en la patogénesis de la hipersensibilidad inmediata a fármacos. Como la inducción de señales de calcio no se traduce necesariamente en una respuesta secretora, parece más significativa la medición de la reacción de desgranulación en el contexto de las pruebas a fármacos (AU)

Immediate moxifloxacin hypersensitivity: Is there more than currently meets the eye?

Immediate drug hypersensitivity reactions (IDHR) to moxifloxacin constitute a pathomechanistic conundrum and a diagnostic challenge. Our objective was to study whether simultaneous phenotyping and quantification of histamine release might add to our knowledge about the basophil activation properties of moxifloxacin and constitute a reliable diagnostic aid. Fifteen patients with an IDHR to moxifloxacin and nine moxifloxacin challenged controls were selected. All had a basophil activation test (BAT) with moxifloxacin. Flow cytometric analysis of basophil responses implied labeling for CD63, CD203c, and intracellular histamine. Unlike tolerant challenged controls, basophilic upregulation of CD203c in response to moxifloxacin was observed in seven of 15 patients. Only two of these seven patients demonstrated appearance of CD63 and release of histamine. In the remainder eight patients, no basophil responses were demonstrable. In conclusion, immediate hypersensitivity to moxifloxacin might involve mechanisms difficult to capture by traditional CD63-/CD203c-based BAT. Deciphering the complexity of quinolone IDHR seems mandatory.

Cannabis sativa allergy: looking through the fog.

IgE-mediated Cannabis (C. sativa, marihuana) allergy seems to be on the rise. Both active and passive exposure to cannabis allergens may trigger a C. sativa sensitization and/or allergy. The clinical presentation of a C. sativa allergy varies from mild to life-threatening reactions and often seems to depend on the route of exposure. In addition, sensitization to cannabis allergens can result in various cross-allergies, mostly for plant foods. This clinical entity, designated as the 'cannabis-fruit/vegetable syndrome', might also imply cross-reactivity with tobacco, natural latex and plant-food-derived alcoholic beverages. Hitherto, these cross-allergies are predominantly reported in Europe and appear mainly to rely upon cross-reactivity between nonspecific lipid transfer proteins or thaumatin-like proteins present in C. sativa and their homologues, ubiquitously distributed throughout plant kingdom. At present, diagnosis of cannabis-related allergies predominantly rests upon a thorough history completed with skin testing using native extracts from crushed buds and leaves. However, quantification of specific IgE antibodies and basophil activation tests can also be helpful to establish correct diagnosis. In the absence of a cure, treatment comprises absolute avoidance measures. Whether avoidance of further use will halt the extension of related cross-allergies remains uncertain.

Phenotypic and functional characterization of in vitro cultured human mast cells.

BACKGROUND: Mast cell progenitor cells, derived from CD34+ hematopoietic stem cells, enter the circulation and subsequently mucosal or connective tissues where they mature to mast cells. Upon activation, mast cells increase the expression of activation markers, e.g. CD63, and release histamine amongst other mediators. Traditionally, release of these mediators is quantified using assays measuring their extracellular concentration in the supernatant of stimulated cells. METHODS: Human mast cells (HuMC) were cultured from peripheral blood, phenotypically characterized, passively sensitized with allogenic IgE antibodies and finally stimulated by anti-IgE that crosslinks IgE/FcεRI complexes. Alterations in the number of cells positive for CD63 and release of histamine were quantified simultaneously by flow cytometry. RESULTS: In culture, two distinct CD45+ cell populations were identified: CD117+ CD203c+hi and CD117- CD203c+low cells. Both populations showed positivity for FcεRI, tryptase and chymase, and contained histamine. Activation resulted in a significant increase of cells positive for CD63+ up to 21% (range: 11-39) for CD117+ CD203c+hi cells (P = 0.005), and 27% (18-55) CD63+ for CD117- CD203c+low cells (P = 0.02). Baseline histamine content was higher for CD117+ CD203c+hi cells than for CD117- CD203c+low cells, respectively 994 (695-6815) Molecules of Equivalent Specific Fluorochrome V500 per cell (MESF-V500/cell) and 797 (629-4978) MESF-V500/cell (P = 0.02). After activation, CD117+ CD203c+hi cells showed significant histamine release of 578 (366-1521) MESF-V500/cell, whilst CD117- CD203c+low cells resulted in 310 (217-366) MESF-V500/cell histamine release. CONCLUSION: This study discloses that culturing HuMC from CD34+ progenitors yields 2 phenotypically distinct cell populations that display a greatly similar response upon cross-linking of IgE/FcεRI complexes. © 2016 International Clinical Cytometry Society.

Shellfish allergens: tropomyosin and beyond.

IgE-mediated shellfish allergy constitutes an important cause of food-related adverse reactions. Shellfish are classified into mollusks and crustaceans, the latter belonging to the class of arthropoda. Among crustaceans, shrimps are the most predominant cause of allergic reactions and thus more extensively studied. Several major and minor allergens have been identified and cloned. Among them, invertebrate tropomyosin, arginine kinase, myosin light chain, sarcoplasmic calcium-binding protein, and hemocyanin are the most relevant. This review summarizes our current knowledge about these allergens.

Quantification of specific IgE antibodies in immediate drug hypersensitivity: More shortcomings than potentials?

BACKGROUND: For many physicians, quantification of serum drug-specific IgE (sIgE) antibodies constitutes the first measure in the diagnostic approach of immediate drug hypersensitivity reactions (IDHR). AIM: To review the accuracy and limitations of the main drug-sIgE tests, especially those that are commercially available. METHODS: A literature search was conducted, using the key-words allergy, diagnosis, drugs, hypersensitivity, specific IgE antibodies; this was complemented by the authors' own experience. RESULTS: The drugs that have mostly been studied appeared to be ß-lactam antibiotics, neuromuscular blocking agents (NMBA) and morphine, the latter as a biomarker for sensitisation to substituted ammonium structures that constitute the major epitope of NMBA. For ß-lactams sensitivity and specificity varied between 0-85% and 52-100%, respectively. For NMBA, sensitivity and specificity varied between 38.5-92% and 92-100%, respectively. With respect to sIgE to morphine it appears this drug to be a sensitive biomarker for sensitisation to rocuronium and suxamethonium but not for atracurium. However, sIgE morphine should not be applied in isolation to diagnose IDHR to NMBA nor opiates. CONCLUSIONS: Although drug-sIgE assay can provide valuable information they should not be performed in isolation to establish correct diagnosis, as their predictive value is not per se absolute. Larger comprehensive studies are urgently required to determine the accuracy of drug-sIgE assays.